Biofuels: The Future for Energy Production?

Question 1

Why is it crucial we obtain a sustainable way of attaining fuel?

Answer 1

It is predicted oil will run out in the next 50-100 years (Stephenson et al. 2010).
The oil that remains is predominantly in the Middle East (66%) , thus the West is concerned about their ‘Energy Secutiy’
Oil is a finite resource, thus must attain a renewable source of energy, specifically energy-dense fuels.

Question 2

What are the first generation of biofuels?

Answer 2

Biodiseal and bioethanol.

Question 3

What is biodiseal and bioethanol?

Answer 3

Biodiseal is derived from storage lipids such as triacylglycerides (TAGs), which can be extracted from oil rich crops such as soybean, rapeseed, jatropha and pal oil. The TAGs are then transesterified to makes FAMEs.
Bioethanol is obtained from sugarcane and cor, sugar and starch are extracted, they undergo hydrolysis and then fermentation to yield ethanol.

Question 4

What are TAGs?

Answer 4

Triacylglycerides - which are transesterified to form fatty acid methyl esters (FAMEs) and glycerol

Question 5

What are FAMEs

Answer 5

Fatty acid methyl esters - which can be separated from glycerol to form biodiseal

Question 6

What are second generation biofuels?

Answer 6

Via enzyme hydrolyssis of lignocellulose of cell walls, to yield pentose sugar which can then be fermented to form ethanol. These are obtained from Misccanthus or willow.

Question 7

What are challenges faced by 2nd generation of biofuels?

Answer 7

The breakdown of lignocellulose to fermentable products, particularly as plant cells have evolved to be resistant to enzymatic degradation.

Question 8

What are the negatives of 2nd generation biofuels?

Answer 8

Require the use of vast amounts of land. Highly energy expensive to chop down trees and transport crops.

Question 9

What are third generation of biofuels?

Answer 9

Biogases

Question 10

What are biogases?

Answer 10

These can be obtained from anaerobic digestion of organic materials, such as waste paper and soft plant tissue, food waste, sewage and animal waste.
These can produce Methane (50-75%), CO2 (20-45%) and Hydrogen (5%).

Question 11

How are biogases created?

Answer 11

Insertion of waste into a anaerobic bioreactor, which digests the waste materials in four main stages:

1. Hydrolysis to produce FAs, AAs and Sugars
2. Acidogenesis to generate Carbonic acids and alcohols, Ammonia, H2 and CO2
3. Acetogenesis to produce Acetic acid, CO2 and H2
4. Methanogenesis –> CH2 and CO2

Question 12

What are the major issue of growing crops for biofuels?

Answer 12

1. Requires huge amounts of fertile land - Soyabeans produce 560 litre/hectare
2. Most land is currently used for agriculture, issue with growing population
3. Life Cycle Analysis (LCA) suggests energy input is greater than output (transport/creation of fertile land)
4. Cultivation required irrigation with fresh water and fertilisers

Question 13

What is algae?

Answer 13

Wide range of simple aquatic organisms that typically carry out oxygenic photosynthesis, can either be macroscopic or microscopic and typically grow in a wide range of environments, particularly environments which would otherwise not be cultivable e.g. hypersaline lakes

Question 14

What are the advantages of algae in the production of biofuels?

Answer 14

1. Rapid Growth Rates - double biomass in hours/days instead of weeks
2. Some species hold extremely high oil content (>50% of dry mass), although hard to extract
3. Manipulate to increase biomass and oil production (dilution of N-free culture)
4. Can be grown without the exploitation of ‘useful’ land
5. Don’t require expensive irrigation
6. Can be linked to CO2 capture

Question 15

How can algae be cultivated?

Answer 15

1. Algal farms/Open ponds
2. Closed photobioreactor (PBRs)
3. Off-shore cultivation

Question 16

What are PBRs?

Answer 16

Photobioreactors; facilitate increased control of growth conditions, e.g. temp, light, CO2 content. However are extremely expensive, harder to scale, problems with O2 build up.

Question 17

What are the adv and neg of Open Ponds?

Answer 17

+ive - cheap, easy to scale
-ive - problems with eco-contamination (GMs), problems with controlling conditions, problems with contamination/competition/predation

Question 18

What is the disadvantage of using algae to generate biofuel?

Answer 18

Biomass ultimately limited by shading problems and photo-inhibition. Thus requires a means of mechanically mixing the system, decreasing overall energy output in LCA, hard to harvest and de-water

Question 19

What methods can be implemented in algal production to improve their production?

Answer 19

1. Flocculation of algal cells - means of aggregation to separate algae from water (de-watering system, often too expensive)
2. Capillary based system - reduce the issue of photo-inhibition
3. Algae Venture Systems Harvester - utilise gravity as the water feed
4. Mutants PSII affecting the antenna - allows for further penetration of light
5. Decrease biosynthesis of starch to increase biomass production and thus TAG production

Question 20

How can light capture be improved?

Answer 20

NB - 95% of light is lost from sunlight.

1. Rapid mixing of culture at 1Hz (energy inefficient)
2. Use of mutants with reduced light-harvesting abilities

Question 21

What mutations can alter light-harvesting abilities?

Answer 21

Those which effect the antenna of the PSII to reduce photoinhibition and allow greater light penetration. Mutants generated following a variant screen. TAM2 - improved by 20% improvement in biomass production.

Question 22

What is photoinhibition?

Answer 22

Photoinhibition is light-induced reduction in the photosynthetic capacity of a plant, alga, or cyanobacterium. Photosystem II (PSII) is more sensitive to light than the rest of the photosynthetic machinery, and most researchers define the term as light-induced damage to PSII.

Question 23

How can we improve TAG production?

Answer 23

1. Starch Biosynthesis

2. Overexpression of key TAG biosynthesis genes during normal growth

Question 24

How does inhibition of starch biosynthesis result in improved TAG synthesis?

Answer 24

Low biomass and high TAG production are both coupled with high starch biosynthesis, induced under nutrient stress. Low starch biosynthesis is additionally coupled with high biomass levels under normal growth conditions. Thus inhibition of starch biosynthesis under nutrient stress allows for a higher biomass production.
Use a simple iodine screen to recognise starch mutants (don’t stain dark blue).

Question 25

Which genes should be overexpressed to result in increased TAG production?

Answer 25

Diacylglycerol acyl transferase (DAGT) - last enzyme in biosynthetic pathway of TAG AcetylCoA carboxylase - the rate limiting step Attempts underway to recognise the 'master' switch that senses nutrient stress and triggers the upregulation of TAG biosynthetic pathway

Question 26

What is the issue with TAG production from algae?

Answer 26

1. Biomass must be harvested and dewatered prior to extraction 2. Cells must be broken and organic solvents used to extract TAGs (energy inefficient) 3. TAGs must be converted to FAMEs (no direct attainment of biofuels)

Question 27

What is the 'ideal algae'

Answer 27

Chlamydomonas reinhardtii

Question 28

Why is C.reinhardtii thought of as the ideal organism?

Answer 28

1. Fast growing species requiring a basic growth medium 2. Generally Recognised as Safe (GRAS) 3. Capable of both phototrophic and heterotrophic growth 4. Genome entirely sequenced 5. Has simple sex cycle 6. Strains can be frozen to prevent genetic drift

Question 29

What can we used as a foreign expression unit?

Answer 29

Chloroplast

Question 30

Why is a chloroplast used as a foreign expression unit?

Answer 30

1. Natural storage compartment allowing for the hyper-accumulation of foreign protein 2. Generation of chloro. transformant is quick 3. Chloroplast genome can be targetted 4. Could express multiple foreign genes as an operon 5. Strong endogenous promoters 6. No gene silencing mechanisms present in the chloroplast

Question 31

How can genes be delivered to the chloroplast?

Answer 31

Shotgun methods - coat gold particles with DNA, shoot at a law of 10(^7) Chlamydomonas, giving rise to 10-100 transformants

Question 32

What are a new line of antibacterials?

Answer 32

Bacteriophages

Question 33

How are mutants with reduced light-harvesting abilities screened?

Answer 33

1. Early log phase culture are subject to 6W/245nm of UV radiation (10% survival) - 20 mins 2. Left in the dark for 2 hours, survivors plated on acetate 3. Undergo fluorescence analysis and chlorophyll a:b analysis 4. Ideal mutants selected following Illumina sequences and mutant analysis

Question 34

How are starch mutants selected for?

Answer 34

1. UV irradiation – to ensure photo-inhibition mutants 2. These were plated onto low N medium 3. Colonies were stained with iodine vapor 4. Re-streak colonies that don’t stain dark blue, thus don’t contain starch as simple carbohydrates oxidize the Iodine to a dark blue color 5. Repeat iodine staining to confirm phenotype 6. Mutants found to accumulate more TAGs

Question 35

What TAG synthesis genes are overexpressed?

Answer 35

1. Diacylglycerol acyl transferase (DAGAT) - the final enzyme in TAG boisynthetic pathway. 2. AcetylCoA Catalase - which is a rate-limiting enzyme (due to low conc.) in the pathway. 3. Attempts have been made to identify the ‘master switch’ that sense N limitation and triggers the up-regulation of the whole TAG biosynthetic pathway

Question 36

What are the issues with using TAGs as biofuels?

Answer 36

1. TAGs require further processing - transesterification to produce FAMEs and a separation protocol to separate glycerol and FAMEs 2. Using algae to make TAGs requires energy expensive processes, such as dewatering 3. Cells must be broken and organic solvents used to extract, again further processing requiring more agents, depletes the total energy obtained from the protocol

Question 37

Describe the integration gene of a chloroplast vector.

Answer 37

Chloroplast transformation vectors are thus designed with homologous flanking sequences on either side of the transgene cassette to facilitate double recombination, which is aided by a RecA type.

Question 38

Describe the insertion site in the chloroplaset genome.

Answer 38

Transcriptionally active spacer regions offer unique advantages, including insertion of transgenes without 5′ or 3′ untranslated regions (UTRs) or promoters. To date, the most commonly used site of integration is the transcriptionally active intergenic region between the trnI-trnA genes, within the rrn operon, located in the IR regions of the chloroplast genome.

Question 39

What is the composition of wood?

Answer 39

Cellulose - Unbranched glucose polymer Hemicellulose - branch polymers composed of glucose plus various hexose and pentose sugars Lignin- complex and heterogenous biopolymer

Question 40

What are other alternative fuels we could obtain?

Answer 40

Fatty alcohols - (C16-18), introduction of plant enzyme FAR might allow precursor fatty acylCoA to be transformed to fatty alcohol. Isoprenoids - are a large family of branched unsaturated carbons. The introduction of isoprene synthase gene (IS) showed isoprene to accumulate in Chlamydomonas. Alkanes - plants make long chain alkanes, some algae and cyanobacteria make using fatty acid reductase (FAR).

Question 41

What gene is deleted during chloroplast transformation?

Answer 41

psbH - small subunit of PSII

Question 42

How are the transformants selected?

Answer 42

delta psbH cells, the expression vector will contain psbH and will recover them to photoautotrophic growth.